To access the open source plugins for Quantitative Autofluorescence or QAF analyses, in the software Fiji. Click on Manage Update sites to add the displayed creative computation update site to the pre-existing update sites. To install the Spectralis pipeline plugin, download the plugins and restart Fiji.
The different Spectralis plugins will be located under the plugins dropdown menu in Spectralis or Spectralis batch. The Spectralis QAF XML export files stored in red, green, blue, or RGB format are limited to a measured AF value scale of zero to 255 and include standard and black calibration regions. To produce a QAF image, open the plugins dropdown menu.
Click Spectralis, select QAF XML reader, and discard the opening screen. A new window showing the prompt, choose a directory containing a spectral XML QAF export will appear. Select the directory and click select.
In the window QAF parameters enter the reference calibration factor or RCF included in the image information, and the patient's age when the image was taken. After clicking okay, when a pop-up labeled map to eight bit appears, enter the minimum QAF or QAF min value, and maximum QAF or QAF max value for a color-coded QAF image. To add one image at a time under plugins, select Spectralis followed by add to standard retina_OCT, and dismiss the opening screen.
When a window displays, choose a directory containing a Spectralis OCT XML export. Select the folder and click select to open the BScan. After the OCT volume loads and the prompt, choose a directory containing registered en Face images appears.
Select the appropriate folder and click select. At this stage, three windows will pop up. One labeled en Face stack, displaying the stacked images from the selected folder.
The second labeled BScan stack, displaying the OCTB scan, and the middle or third one labeled choose modality. Select a modality from the en Face stack. As a new window with the prompt, choose a directory containing the standard retina pops up, select or create the directory containing standard retina.
Examine the new standard retina, scroll and move the cursor to view the mean, and standard deviation for that specific location. Click on the button Accept, to either add the latest photo to the standard retina, or discard it. To add multiple images at a time using Batch_QAF_ standard retina first, prepare a manifest.
txt file in the same folder as the case IDs. The relative path from the location of the dot txt file to the OCT and the enFace stack must be listed, and separated by a tab space without any additional white space before and after the names. To scroll through the BScan stack, drag the bar at the bottom to the left or right, or select the BScan frame, and use the left and right arrow keys on the keyboard.
An overview of the current BScan stack area, is provided by the red line on the enFace stack window, and on the top left of the BScan window where the BScan number is displayed. To mark a region, select mark in the draw line segment in BScan window, and start by right clicking and dragging the mouse cursor to the end of the lesion. Press the plus key to zoom in, and the minus key to zoom out.
Finally, click okay to mark the region of interest. The QAF image of an eye of an 84 year old patient with intermediate age related macular degeneration, showed a singular marked soft drusen. A closeup of the area showed a brown center representing the marked drusen, and colored bands representing the surrounding ISO holes.
The output file contained the case ID, the laterality of the file, and the chosen imaging modality. Each row referred to an ISO hole, whose distances from the outer edge of the lesion were specified in the lower, and upper columns of the spreadsheet. The QAF measurements contained the minimum median, and maximum values of a pixel in an ISO hole, the number of pixels, the mean pixel value, and the standard deviation of the mean.
The QAF image of an eye of an 80 year old patient with early age-related macular degeneration, showed subretinal, drusenoid deposits. Around each marked lesion, the ISO holes were depicted with color coding.
